Chromosomal fragile sites are specific loci which are especially susceptible to forming breaks or rearrangements when cells are cultured under conditions that inhibit DNA replication. The FRA3B at 3p14.2, lies within the FHIT gene, and is the most highly expressed of the common fragile sites observed when DNA replication is perturbed by aphidicolin, an inhibitor of DNA polymerase a/d. Common fragile sites are highly unstable regions of the genome. Large intragenic deletions within FHIT, localized within the FRA3B sequences, have been identified in a variety of tumor cells. In the current funding period, we have begun to examine the mechanism for fragility at FRA3B. During the past three years, we demonstrated that the FRA3B replicates late in S phase, we mapped the late replicating domain containing the FRA3B and we demonstrated that this domain correlates with the genetically unstable sequences. In addition, we have mapped the deletions occurring within FRA3B in a large panel of chromosome 3 homologues from tumor cells, and have identified the sequences required for instability.The molecular basis for instability at common fragile sites is unknown, but induction appears to involve the interruption of normal DNA synthesis. We have hypothesized that fragile sites are regions of DNA whose replication is unusually sensitive to interference, and that the visible chromosome breaks and gaps are due to unreplicated DNA. The major goal of this proposal is to determine the mechanism(s) of fragile site expression and genetic instability. Specifically, we propose (1) to elucidate the role of DNA replication in instability at fragile sites (Aim 1); (2) to determine whether cell cycle checkpoints are activated as a consequence of fragile site induction (Aim 2); and (3) to determine which DNA repair pathways are involved in repairing DNA damage at fragile sites (Aim 3). Genomic instability is a frequent characteristic of tumor cells, and common fragile sites may be particularly sensitive to such instability. We believe that the mechanisms involved in rearrangements at fragile sites are likely to apply to other sites of rearrangements in cancer; thus, our studies may lead to important insights into the pathogenesis of human tumors.